T lymphocytes play a critical role in host defense mechanisms and in autoimmunity or transplant rejection. The T cell response is a highly regulated process. Activation of resting T cells is initiated by cell surface molecules and results in the generation of intracellular second messengers that influence a variety of cellular processes which lead to clonal differentiation and proliferation. A detailed understanding of the molecular events regulating the activation of T cells is critical towards our ability to manipulate the T cell response. The principle molecule regulating-the activation of T cells by antigen is the T cell antigen receptor complex (TCR), composed of the products of six genes. Although the TCR has no intrinsic enzymatic functions, stimulation of the receptor activates intracellular signaling molecules including protein tyrosine kinases (PTKs) and phospholipase C (PLC). The mechanism by which the TCR activates these functions is not clear, nor are the components of the signaling pathway leading to the activation of PLC. The initial five year grant proposed to use a somatic cell genetic approach to identify gene products involved in the TCR signal transduction pathway by isolating signaling deficient mutants of a T cell line. These studies led to the identification of four complementation groups defining components of the signal transduction pathway. Two of these groups represent the products of two defined genes, lck and CD45. Lck is a protein tyrosine kinase of the src family and CD45 is a transmembrane tyrosine phosphatase that is likely to regulate lck activity. This renewal proposes to utilize the isolated mutant cell lines as well as a novel methodology utilizing tyrosine phosphopeptides to study the role of lck and CD45 in regulating TCR signal transduction. The mutant cells deficient in lck or CD45 will be reconstituted with mutant, chimeric or other PTKs or PTPase in order to define the role of these molecules. These genetic reconstitution experiments will be supplemented with biochemical analyses. The goal of these studies is to understand: 1) the functions of lck and CD45; 2) how their functions are regulated; and, 3) with which molecules they interact in the signaling pathway.